Examination of adenylate cyclase in plasma membrane preparations from rat adipocytes, brain cortex, cordate nucleus and human platelets revealed that activity is inhibition by putative neurotransmitters. Depending on the cell from which membranes were derived, inhibition was seen with adenosine, epinephrine, prostaglandins and opiates. As with stimulation of cyclases by hormones and neurotransmitters, inhibition was dependent on GTP. However, in contrast to stimulatory hormones, inhibition was: 1) enhanced by sodium and lithium ions, and 2) not expressed in the presence of Gpp(NH)p. Moreover, chemical treatments of membranes selectively destroyed the effects of stimulatory hormones, but not of inhibitory hormones, and vice versa. Finally, target size analysis indicated that the GTP-dependent stimulatory and inhibitory actions are mediated by structures of different size. It is concluded that dual regulation (stimulation and inhibition) by receptor-mediated processes is a wide-spread feature of adenylate cyclase.